Abstract
The regulation of human Arf1 GTPase activity by ArfGEFs that stimulate GDP/GTP exchange and ArfGAPs that mediate GTP hydrolysis has attracted attention for the discovery of Arf1 inhibitors as potential anti-cancer agents. The malaria parasite Plasmodium falciparum encodes a Sec7 domain-containing protein - presumably an ArfGEF - and two putative ArfGAPs, as well as an Arf1 homologue (PfArf1) that is essential for blood-stage parasite viability. However, ArfGEF and ArfGAP-mediated activation/deactivation of PfArf1 has not been demonstrated. In this study, we established an in vitro colorimetric microtiter plate-based assay to detect the activation status of truncated human and P. falciparum Arf1 and used it to demonstrate the activation of both proteins by the Sec7 domain of ARNO, their deactivation by the GAP domain of human ArfGAP1 and the inhibition of the respective reactions by the compounds SecinH3 and QS11. In addition, we found that the GAP domains of both P. falciparum ArfGAPs have activities equivalent to that of human ArfGAP1, but are insensitive to QS11. Library screening identified a novel inhibitor which selectively inhibits one of the P. falciparum GAP domains (IC50 4.7 µM), suggesting that the assay format is suitable for screening compound collections for inhibitors of Arf1 regulatory proteins.
Highlights
ADP-ribosylation factor (Arf) GTPases are central regulators of protein trafficking in eukaryotic cells
We focused on the two sequences which are annotated as ArfGAPs on the plasmodb.org malaria genome database, which we designated as PfArfGAP1 (Plasmodb entry PF3D7_1244600) and PfArfGAP2 (PF3D7_0526200.1)
We focused on the GTPase activating proteins (GAPs) domain of PfArfGAP1 since, in contrast to PfArfGAP2, the coding sequence has been reported to be essential to the survival of blood stage P. falciparum and P. berghei parasites in genome-wide knockout and transposon mutagenesis studies[39,40]
Summary
ADP-ribosylation factor (Arf) GTPases are central regulators of protein trafficking in eukaryotic cells. Identified by probing a P. falciparum genomic library and PCR from P. falciparum cDNA23–25, the recombinant protein was shown to bind GTP, have ADP-ribosyltransferase and phospholipase D stimulating activity in addition to low intrinsic GTPase activity, all features of Arf GTPases[24,25] It is capable of stimulating P. falciparum phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which is an established role of mammalian Arf[1] in the regulation of phosphorylated phosphatidylinositol levels and, membrane trafficking, signalling and cytoskeleton dynamics[26]. In this study, using human recombinant proteins as a model, we developed a novel microtiter plate-based assay to detect Arf[1] activation (GTP vs GDP-bound) status and modulation of it by an ArfGEF (ARNO) Sec[7] domain and Arf GAP (ArfGAP1) GAP domain. Given the interest in Arf[1] as a drug target, a further motivation for developing the assay was to introduce an assay format compatible with the screening of compound libraries for Arf[1] activity modulators, explored here by detecting the differential inhibition of ARNO and GAP-mediated Arf[1] activation/deactivation using standard inhibitors, as well as the identification of a novel, selective PfArf[1] GAP inhibitor
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
